ABSTRACT A well informed experimental genetic cross is a powerful forward genetics tool because it generates recombinant progeny for genetic mapping of observed phenotypes. This approach has been extensively used in many model organisms from flies to mice but crossing different strains of the human malaria parasite Plasmodium falciparum, responsible for the deaths of hundreds of thousands of individuals every year, is a challenge. The parasite's life cycle is complex, alternates between the mosquito vector and human host and includes obligate sexual reproduction. In order to generate and phenotype recombinant parasites, the complete life cycle from asexual blood stage-to-sexual blood stage-to-recombinant formation in the mosquito-to-liver stage and back to asexual blood stage must occur in the laboratory. Previously, the liver stage-to blood stage transition was only possible in splenectomized chimps and due to the ethical and financial roadblocks to chimp research only three genetic crosses were ever performed before the NIH banned chimp research. We have developed a human-liver chimeric mouse model that allows for P. falciparum liver stage development and transition to blood stage infection and have used this model successfully to recover progeny from P. falciparum genetic crosses. The ease of use of this mouse model will now allow us to create numerous well-informed crosses. This Research Project will initially define variables and factors that will allow us to maximize the recovery of unique recombinant parasites. We predict that with >60 unique recombinant progeny, we will be able to fine map traits of interest to genome regions containing just a few candidate genes. Furthermore, we will use recombinant progeny for backcrossing experiments to determine genes involved in the parasite's ability to avoid selfing and promote hybridization as well as cytoplasmic genome incompatibility observed in crossing experiments. Accelerated hybridization could drive recombination between parasite strains and thus could speed the spread of drug resistant genes through a parasite population. Conversely, cytoplasmic genome incompatibility could prevent parasites strains from recombining. The mating mechanisms analyzed within this project are of great importance in the context of the overall P01 grant, which aims to more fully understand artemisinin resistance and resistance to partner drugs, including piperaquine, which currently are a significant concern in malaria elimination efforts. Finally, we will establish bulk segregant analyses coupled with genome sequencing and quantitative trait loci mapping to enable more efficient mapping of genes linked to phenotypes and this will be especially important for analysis of emerging piperaquine resistant parasites that have now been documented in Southeast Asia. The successful completion of this Research Project will greatly enhance P. falciparum genetics research and will further our understanding of parasite drug resistance, with regard to its emergence, evolution and spread.